Immunoglobulins (antibodies) are physiologically active substances that are responsible for immune response. In recent years, their use values have been increasing in applications such as pharmaceutical products, diagnostic drugs, and materials for separation and purification of corresponding antigenic proteins. The antibodies are obtained from the blood of immunized animals, cell culture solutions of cells possessing the ability to produce antibodies, or ascitic fluid culture solutions of animals. However, such antibody-containing blood or culture solutions contain proteins other than the antibodies, or complicated foreign components derived from stock solutions used in the cell culture. The separation and purification of the antibodies from these impurity components usually require a complicated and time-consuming operation.
Liquid chromatography is important for antibody separation and purification. Examples of chromatography approaches for antibody separation include gel filtration chromatography, affinity chromatography, ion-exchange chromatography, and reverse-phase chromatography. Antibodies are separated and purified by combining these approaches.
The ion-exchange chromatography is a method which involves using ion-exchange groups on the surface of an adsorbent as a stationary phase and reversibly adsorbing thereon counterions present in a mobile phase for separation. For example, beads or a membrane (e.g., flat membranes and hollow fiber) is adopted as the shape of the adsorbent. These matrices bound with cation-exchange groups or anion-exchange groups are commercially available as adsorbents.
Purification using the adsorbent having cation-exchange groups is generally performed by contacting an antibody solution having a low salt concentration with the adsorbent so that antibodies are adsorbed thereon, and eluting the adsorbed physiologically active substances by increasing the salt concentration of a mobile phase. The purification of a substance of interest in a flow-through manner described below has also been proposed as a more favorable method.
The flow-through manner is a manner of a purification method that selectively adsorbs impurities rather than the substance of interest onto an adsorbent. Therefore, this approach leads to a saving in buffer solution and simplification of steps, as compared with conventional methods using adsorption and elution. Also, it is considered that the advantages of the flow-through purification can be further exploited if an antibody solution can be processed at a high flow rate.
A cation-exchange step is often aimed at separating antibody monomers from aggregates such as antibody dimers. However, the antibody monomers and the antibody aggregates have almost equal isoelectric points. Therefore, the separation of antibody monomers from antibody dimers is particularly difficult for the flow-through purification and requires the detailed design of a cation-exchanger, including the density of cation-exchange groups. In addition, each antibody has distinctive properties. Therefore, the same design is not always optimal for all antibodies even if the design is detailed. It is considered that there is a suitable cation-exchange group density for each antibody.
Patent Literature 1 discloses a multimodal chromatographic resin for use in flow-through purification, comprising a resin bound with low-molecular compounds having aromatic and weak cation-exchange groups.
Patent Literature 2 discloses a chromatographic support comprising silica beads bound with copolymers having weak cation-exchange groups.
Patent Literature 3 discloses a chromatographic support suitable for flow-through purification in which monomers having strong cation-exchange groups and uncharged (neutral) monomers are graft-polymerized onto a support.